Maltotriosyl transferase, process for production thereof, and use thereof

ABSTRACT

The object is to provide a novel glycosyltransferase and the use thereof, the glycosyltransferase catalyzes transglucosylation of maltotriose units under conditions which can be employed for the processing of foods or the like. Provided is a maltotriosyl transferase which acts on polysaccharides and oligosaccharides having α-1,4 glucoside bonds, and has activity for transferring maltotriose units to saccharides, the maltotriosyl transferase acting on maltotetraose as substrate to give a ratio between the maltoheptaose production rate and maltotriose production rate of 9:1 to 10:0 at any substrate concentration ranging from 0.67 to 70% (w/v).

TECHNICAL FIELD

The present invention relates to a maltotriosyl transferase and the usethereof, and specifically to a novel maltotriosyl transferase and amethod for producing the same, the use of the enzyme in food productionand processing, and a microorganism producing the enzyme. The presentapplication claims priority based on Japanese Patent Application No.2009-156569 filed on Jul. 1, 2009, and the content of the patentapplication is hereby incorporated by reference herein in its entirety.

BACKGROUND ART

Maltotriose-producing amylases heretofore known are the enzymes derivedfrom Microbacterium imperiale, Streptomyces griseus, Bacillus subtilis,Natronococcus sp., and Streptococcus bovis (Non-Patent Document 1).However, among these enzymes, only the Streptomyces griseus-derivedenzyme is reported about its involvement with transglucosylation. Inaddition, this enzyme catalyzes transglucosylation only when thesubstrate concentration is high (the sum of the donor and acceptorsubstrates is 19%, 40% (w/v)), while catalyzes hydrolysis reaction alonewhen the substrate concentration is low (1% (w/v)), and will notcatalyze transglucosylation (Non-Patent Documents 2 and 3). In addition,the enzyme is poorly resistant to heat, and thus is not used for foodprocessing purposes.

Examples of industrially used glycosyltransferase include α-glucosidaseused for the production of isomaltooligosaccharide ornigerooligosaccharide, β-fructofuranosidase used for the production offructo-oligosaccharide or lactosucrose, β-galactosidase used for theproduction of galactooligosaccharide, α-glucosyltransferase used for theproduction of palatinose, cyclodextringlucanotransferase used for theproduction of cyclodextrin or coupling sugar, and branching enzymes usedfor the production of highly branched cyclic dextrin. Among these,α-glucosidase and branching enzymes act on polysaccharides andoligosaccharides containing α-1,4 bonds to catalyze transglucosylation.α-glucosidase catalyzes transglucosylation of monosaccharides, andbranching enzymes catalyze transglucosylation of oligosaccharidescontaining four or more sugars or polysaccharides. There is no knownenzyme which specifically catalyzes transglucosylation of maltotriosewhich is a trisaccharide.

In processed food containing starch, retrogradation of starch causesserious problems such as deterioration of storage stability.Retrogradation of starch is caused mainly by retrogradation of amylosemolecules contained in starch, more specifically association of amylosemolecules accompanied by insolubilization (Non-Patent Document 4). As aresult of research on retrogradation control through starchdepolymerization, retrogradation control is now possible to some degree.However, such depolymerized starch loses its intrinsic properties. Inaddition, decomposed starch has higher reducing power, and thus canreact with a protein or amino acid when heated together, which resultsin coloring of the starch. Therefore, these methods have found limitedapplications (Patent Document 1). In order to solve these problems,studies for controlling retrogradation of starch withoutdepolymerization have been carried out. For example, branching enzymeswhich decompose α-1,4 bonds of starch and synthesize α-1,6 bonds bytransfer reaction are studied, but they have poor heat resistance, andthus are not used as food processing enzymes.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: Japanese Unexamined Patent Application    Publication No. 2001-294601

Non-Patent Documents

-   Non-Patent Document 1: “Denpun Kagakuno Jiten”, Asakura Publishing    Co., Ltd., p. 279-80 (2003) Non-Patent Document 2: Wakao et al,    Journal of the Japanese Society of Starch Science, 25(2), p.    155-61 (1978) Non-Patent Document 3: Usui et al, Carbohydr. Res.    250, 57-66 (1993) Non-Patent Document 4: Okada et al, Journal of the    Japanese Society of Starch Science, 30(2), p. 223-230 (1983)    Non-Patent Document 5: Saito, and Miura. Biochim. Biophys. Acta, 72,    619-629 (1963)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention is intended to provide a novel glycosyltransferasewhich catalyzes transglucosylation of maltotriose units under conditionswhich can be employed for the processing of foods or the like.

Means for Solving Problem

The inventors diligently made researches for solving the above-describedproblems. As a result of this, they have found that a microorganismbelonging to the genus Geobacillus produces a maltotriosyl transferasewhich has the desired function. Furthermore, the inventors isolated andpurified the maltotriosyl transferase, and have succeeded in determiningthe enzymatic chemical properties, and cloning the gene coding theenzyme (hereinafter referred to as, “the present gene”). In addition,they established the method for producing a maltotriosyl transferasethrough the introduction of the present gene and fragments of thepresent gene into an appropriate host. The present invention has beenaccomplished based on these results, and its aspects are as follows. [1]A maltotriosyl transferase which acts on polysaccharides andoligosaccharides having α-1,4 glucoside bonds to transfer maltotrioseunits to saccharides, the maltotriosyl transferase acting onmaltotetraose as substrate to give a ratio between the maltoheptaoseproduction rate and maltotriose production rate of 9:1 to 10:0 at anysubstrate concentration ranging from 0.67 to 70% (w/v). [2] Themaltotriosyl transferase according to [1], wherein the maltotriosyltransferase is an enzyme derived from a microorganism. [3] Themaltotriosyl transferase according to [1], wherein the maltotriosyltransferase is an enzyme derived from a microorganism belonging to thegenus Geobacillus. [4] The maltotriosyl transferase according to [3],wherein the microorganism belonging to the genus Geobacillus isGeobacillus sp. APC9669 (accession number NITE BP-770). [5] Amaltotriosyl transferase comprising the following enzymatic chemicalproperties:

(1) action: acts on polysaccharides and oligosaccharides having α-1,4glucoside bonds as a binding mode to transfer maltotriose units tosaccharides;

(2) substrate specificity: acts on soluble starch, amylose, amylopectin,maltotetraose, maltopentaose, and maltohexaose, while does not act onα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, maltotriose, andmaltose; and

(3) molecular weight: about 83,000 (SDS-PAGE). [6] An enzyme productcomprising the maltotriosyl transferase of any one of [1] to [5] as anactive ingredient. [7] A microorganism having a capability to producemaltotriosyl transferase, the microorganism being Geobacillus sp.APC9669 (accession number NITE BP-770) or its mutant strain. [8] Amaltotriosyl transferase comprising the amino acid sequence set forth inSEQ ID NO: 8, or its fragment exhibiting maltotriosyl transferaseactivity. [9] The maltotriosyl transferase according to [8] coded by aDNA comprising the sequence set forth in SEQ ID NO: 6. [10] Amaltotriosyl transferase gene comprising any of the DNAs selected fromthe group consisting of the following (a) to (e): (a) DNA coding theamino acid sequence set forth in SEQ ID NO: 7 or 8; (b) DNA comprisingthe sequence set forth in SEQ ID NO: 6; (c) DNA hybridizing with thecomplementary sequence of the sequence set forth in SEQ ID NO: 6 understringent conditions; (d) DNA which is a degenerate of the DNA sequenceof the sequence set forth in SEQ ID NO: 6; (e) DNA coding comprising asequence including substitution, deletion, insertion, addition, orinversion of one or a plurality of bases with reference to the sequenceset forth in SEQ ID NO: 6, and coding a protein having maltotriosyltransferase activity. [11] A recombinant vector comprising themaltotriosyl transferase gene of [10]. [12] The recombinant vector of[11], which is an expression vector. [13] A transformant into which themaltotriosyl transferase gene of [10] has been introduced. [14] Atransformant into which the recombinant vector of [11] or [12] has beenintroduced.

[15] The transformant according to [13] or [14], which is a bacterialcell, a yeast cell, or a fungal cell. [16] A method for producing amaltotriosyl transferase, comprising the following steps (1) and (2), orthe steps (i) and (ii): (1) culturing a microorganism belonging to thegenus Geobacillus having a capability to produce a maltotriosyltransferase; and (2) collecting the maltotriosyl transferase from theculture solution and/or bacterial cells after culturing. (i) culturingthe transformant of any one of [13] to [15] under conditions suitablefor the production of the protein coded by the maltotriosyl transferasegene; and (ii) collecting the protein thus produced. [17] The productionmethod according to [16], wherein the microorganism belonging to thegenus Geobacillus is Geobacillus sp. APC9669. [18] A use of the enzymeof any one of [1] to [5], or the enzyme product of [6] for producing andprocessing a food containing a polysaccharide or oligosaccharide havingα-1,4 glucoside bonds. [19] A food or food ingredient which has beenimproved in its function by the use of the enzyme of any one of [1] to[5], or the enzyme product of [6].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the optimum temperature for the maltotriosyltransferase derived from Geobacillus sp. APC9669.

FIG. 2 is a graph showing the optimum pH for the maltotriosyltransferase derived from Geobacillus sp. APC9669.

FIG. 3 is a graph showing the thermostability of the maltotriosyltransferase derived from Geobacillus sp. APC9669.

FIG. 4 is a graph showing the pH stability of the maltotriosyltransferase derived from Geobacillus sp. APC9669.

FIG. 5 shows the result of SDS-PAGE analysis on the maltotriosyltransferase. Lane 1: molecular weight marker; Lane 2: maltotriosyltransferase.

FIG. 6 shows the results of bread softness retention test.

FIG. 7 shows the results of SDS-PAGE on the centrifugation supernatantsof cell fragments from an Escherichia coli transformant. Lane M:molecular weight marker; Lane 1: centrifugation supernatant of cellfragments from E. coli vector transformant; Lane 2: maltotriosyltransferase.

DETAILED DESCRIPTION OF THE INVENTION

(Term) In the present invention, the term “DNA coding a protein” refersto the DNA which gives the protein upon expression, more specificallythe DNA having a base sequence corresponding to the amino acid sequenceof the protein. Accordingly, codon degeneracy is taken intoconsideration.

In the present description, the term “isolated” may be replaced with“purified”. When the enzyme of the present invention (maltotriosyltransferase) is derived from a natural material, the term “isolated”used for the enzyme means that the enzyme is substantially free ofcomponents of the natural material other than the enzyme (specificallysubstantially free of contaminant protein). Specifically, for example,in the isolated enzyme of the present invention, the content ofcontaminant proteins is less than about 20%, preferably less than about10%, more preferably less than about 5%, and even more preferably lessthan about 1% in the weight equivalence. On the other hand, when theenzyme of the present invention is prepared by a genetic engineeringtechnique, the term “isolated” means that the enzyme is substantiallyfree of other components derived from the host cells or culture solutionused. Specifically, for example, in the isolated enzyme of the presentinvention, the content of contaminant components is less than about 20%,preferably less than about 10%, more preferably less than about 5%, andeven more preferably less than about 1% in the weight equivalence. Inthe present description, the simple term “maltotriosyl transferase”means “isolated maltotriosyl transferase”, unless it is evident that theterm has a different meaning. The same applies to the term “the presentenzyme” used in place of maltotriosyl transferase.

When the term “isolated” is used for a native DNA, the term typicallymeans that the DNA is separated from other nucleic acids coexisting withthe DNA in its natural state. However, the DNA may contain some of othernucleic acid components, such as a flanking nucleic acid sequence of theDNA in its natural state (for example, the sequence of the promoterregion or the terminator sequence). For example, when a genome DNA is inan “isolated” state, it is preferably substantially free of other DNAcomponents which coexist with the DNA in its natural state. On the otherhand, when a DNA prepared by a genetic engineering technique, such as acDNA molecule, is in an “isolated” state, it is preferably substantiallyfree of cell components or culture solution. In addition, when a DNAprepared by chemical synthesis is in an “isolated” state, it ispreferably substantially free of precursors (raw materials) such as dNTPor chemical substances used during synthesis. In the presentdescription, the simple term “DNA” means an isolated DNA, unless it isevident that the term has a different meaning.

(Maltotriosyl transferase and bacterium producing the same) A firstaspect of the present invention is to provide a maltotriosyl transferase(hereinafter, also referred to as “the present enzyme”) and a bacteriumproducing the same. As described in the below-described Examples, theinventors carried out dedicated research, and have found thatGeobacillus sp. APC9669 produces a maltotriosyl transferase.Furthermore, they have succeeded in isolating and producing themaltotriosyl transferase, and, as described below, in determining theenzymatic chemical properties of the enzyme.

(1) Action The present enzyme is a maltotriosyl transferase, and acts onpolysaccharides and oligosaccharides having α-1,4 glucoside bonds as abinding mode to transfer maltotriose units to saccharides.

(2) Substrate specificity The present enzyme favorably acts on solublestarch, amylose, amylopectin, maltotetraose, maltopentaose, andmaltohexaose. On the other hand, the present enzyme does not act onα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, maltotriose, andmaltose.

(3) Molecular weight The molecular weight of the present enzyme is about83,000 (by SDS-PAGE).

(4) Optimum temperature The optimum temperature for the present enzymeis about 50° C. The present enzyme exhibits high activity in thetemperature range of about 45° C. to 55° C. The optimum temperature wascalculated by the below-described method for measuring maltotriosyltransferase activity (in 10 mmol/L MES buffer solution (pH 6.5)).

(5) Optimum pH The optimum pH for the present enzyme is about 7.5. Thepresent enzyme exhibits high activity in the pH range of about 6.5 to8.0. The optimum pH is determined based on, for example, the measurementin a universal buffer solution.

(6) Thermostability The present enzyme exhibits stable activity at 65°C. or lower. The present enzyme keeps 90% or higher level of activityeven after treatment for 30 minutes at 65° C. in a 10 mmol/L MES buffersolution (pH 6.5).

(7) pH stability The present enzyme exhibits stable activity in a widepH range of 5.0 to 10.0. More specifically, the enzyme keeps 85% orhigher level of activity after treatment for 30 minutes at 40° C., aslong as the pH of the enzyme solution used for the treatment is withinthe above range.

(8) Isoelectric point The isoelectric point of the present enzyme isabout 4.5 (by Ampholine electrophoresis).

As shown in the below-described Examples, when the maltotriosyltransferase produced by Geobacillus sp. APC9669 acts on maltotetraose assubstrate, it gives a ratio between the maltoheptaose production rateand maltotriose production rate of 9:1 to 10:0 at any substrateconcentration ranging from 0.67 to 70% (w/v), wherein maltoheptaose is atransglycosylation product and maltotriose is a decomposition product,respectively. In other words, the rate of transglucosylation is farhigher over the wide substrate concentration range, and themaltoheptaose production rate was 90% or more, taking the sum of themaltoheptaose and maltotriose production rates as 100%. The rates werecompared based on the molar ratios of the products.

As described above, details about the properties of the present enzymeobtained herein have been revealed. As a result of this, it has beenfound that the present enzyme has high heat resistance and highsubstrate specificity. Accordingly, the present enzyme is suitable forfood processing.

The present enzyme is preferably a maltotriosyl transferase derived fromGeobacillus sp. APC9669. The term “maltotriosyl transferase derived fromGeobacillus sp. APC9669” in this case means a maltotriosyl transferaseproduced by Geobacillus sp. APC9669 (wild strain or mutant strain), or amaltotriosyl transferase obtained by a genetic engineering techniqueusing the maltotriosyl transferase gene of Geobacillus sp. APC9669 (wildstrain or mutant strain). Accordingly, “maltotriosyl transferase derivedfrom Geobacillus sp. APC9669” includes the recombinants produced by hostmicroorganisms into which the maltotriosyl transferase gene (or themodified version of the gene) obtained from Geobacillus sp. APC9669,“Geobacillus sp. APC9669 has been introduced.

For convenience of explanation, Geobacillus sp. APC9669 which is thesource of the present enzyme is referred herein the bacterium producingthe present enzyme. The APC9669 strain is deposited on thebelow-described depository, and is readily available therefrom.Depository institution: Patent Microorganisms Depository, NITEBiotechnology Development Center (2-5-8, Kazusakamatari, Kisarazu-shi,Chiba, 292-0818, Japan) Date of deposit (date of receipt): Jun. 2, 2009

Accession number: NITE BP-770

The maltotriosyl transferase of the present invention according to oneembodiment contains the amino acid sequence set forth in SEQ ID NO: 8.This amino acid sequence is the amino acid sequence set forth in SEQ IDNO: 7 excluding the signal peptide portion. The amino acid sequence setforth in SEQ ID NO: 7 was deduced from the base sequence (SEQ ID NO: 6)of the gene obtained by cloning Geobacillus sp. APC9669. In general,when the amino acid sequence of a certain protein is partially modified,the modified protein may have equivalent function to the unmodifiedprotein. More specifically, modification of the amino acid sequence doesnot substantially influence the function of the protein, and thus thefunction of the protein may be maintained before and after themodification. Accordingly, another embodiment of the present inventionprovides a protein which is composed of an amino acid sequenceequivalent to the amino acid sequence set forth in SEQ ID NO: 8, and hasmaltotriosyl transferase activity (hereinafter, also referred to as“equivalent protein”). The term “equivalent amino acid sequence” in thiscase means an amino acid sequence which is partially different from theamino acid sequence set forth in SEQ ID NO: 8, but the difference doesnot substantially influence the function of the protein (maltotriosyltransferase activity). The term “maltotriosyl transferase activity”means the activity for polysaccharides and oligosaccharides having α-1,4glucoside bonds as a binding mode to transfer the maltotriose units tosaccharides. The degree of the activity is not particularly limited aslong as the function of a maltotriosyl transferase can be exhibited, butis preferably equivalent to or higher than that of the protein composedof the amino acid sequence set forth in SEQ ID NO: 8.

The term “partial difference in the amino acid sequence” typically meansmutation (change) in the amino acid sequence caused by deletion orsubstitution of one to several (up to, for example, 3, 5, 7, or 10)amino acids composing the amino acid sequence, or addition, insertion,or combination thereof of one to several (up to, for example, 3, 5, 7,or 10) amino acids. The difference in the amino acid sequence isacceptable as long as the maltotriosyl transferase activity ismaintained (the activity may be varied to a degree). As long as theconditions are satisfied, the position of the difference in the aminoacid sequence is not particularly limited, and the difference may arisein a plurality of positions. The term “plurality” means, for example, anumber corresponding to less than about 30%, preferably less than about20%, more preferably less than about 10%, even more preferably less thanabout 5% of the total amino acids, and most preferably less than about1%. More specifically, the equivalent protein has, for example, about70% or more, preferably about 80% or more, even more preferably about90% or more, even more preferably about 95% or more, and most preferablyabout 99% or more identity with the amino acid sequence set forth in SEQID NO: 8.

Preferably, the equivalence protein is obtained by causing conservativeamino acid substitution in an amino acid residue which is not essentialfor maltotriosyl transferase activity. The term “conservative amino acidsubstitution” means the substitution of an amino acid residue withanother amino acid residue having a side chain with similar properties.Amino acid residues are classified into several families according totheir side chains, such as basic side chains (for example, ricin,arginine, and histidine), acidic side chains (for example, aspartic acidand glutamic acid), uncharged polar side chains (for example, glycine,asparagine, glutamine, serine, threonine, tyrosine, and cysteine),nonpolar side chains (for example, alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, and tryptophan), β branched sidechains (for example, threonine, valine, and isoleucine), and aromaticside chains (for example, tyrosine, phenylalanine, tryptophan, andhistidine). Conservative amino acid substitution is preferably thesubstitution between amino acid residues in one family.

The “equivalent protein” may have additional properties. For example,the equivalent protein may have higher stability than the proteincomposed of the amino acid sequence set forth in SEQ ID NO: 8, mayperform a different function performed only at low temperatures and/orhigh temperatures, or may have a different optimum pH.

The identity (%) between the two amino acid sequences may be determinedby, for example, the following procedure. Firstly, the two sequences arealigned for optimal comparison (for example, a gap may be introducedinto the first sequence thereby optimizing the alignment with the secondsequence). When the molecule at the specific position in the firstsequence (amino acid residue) is the same as the molecule at thecorresponding position in the second sequence, the molecules at thepositions are regarded as identical. The sequence identity is thefunction of the number of the identical positions common to the twosequences (more specifically, identity (%)=number of identicalpositions/total number of positions×100), and preferably the number andsize of the gaps required for the optimization of the alignment aretaken into consideration. Comparison of the two sequences anddetermination of the identity are achieved using a mathematic algorithm.Specific examples of the mathematic algorithm usable for the comparisonof the sequences include, but not limited to, the algorithm described inKarlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, andmodified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-77. These algorithms are incorporated into NBLAST Program andXBLAST Program (version 2.0) described in Altschul et al. (1990) J. Mol.Biol. 215:403-10. For example, under XBLAST Program, when BLASTpolypeptide is searched under conditions that score=50 and wordlength=3,an amino acid sequence with a high identity can be obtained. GappedBLAST described in Altschul et al. (1997), Amino Acids Research 25(17):3389-3402 can be used for obtaining a gap alignment for comparison. WhenBLAST and Gapped BLAST are used, default parameters of correspondingprograms (for example, XBLAST and NBLAST) may be used. For more detailedinformation, see, for example, the website of NCBI. Other examples ofthe mathematic algorithm usable for the comparison of the sequencesinclude the algorithm described in Myersand Miller (1988) Comput ApplBioSci. 4: 11-17. These algorithms are incorporated into, for example,ALIGN Program usable in GENESTREAM Network Server (IGH Montpellier,France) or ISREC Server. When ALIGN program is used for the comparisonof amino acid sequences, for example, a PAM120 weight residue table isused, wherein the gap length penalty is 12, and the gap penalty is 4.The identity between the two amino acid sequences is determinedaccording to GAP Program of GCG software package using Blossom 62 matrixor PAM250 matrix, wherein the gap weight is 12, 10, 8, 6, or 4, and thegap length weight is =2, 3, or 4.

The present enzyme may be a portion of a larger protein (for example, afused protein). Examples of the sequence added to a fused proteininclude the sequences useful for purification of multiple histidineresidues, and addition sequences which ensures stability inrecombination production.

The present enzyme having the above-described amino acid sequence isreadily prepared by a genetic engineering technique. For example, anappropriate host cell (for example, Escherichia coli) is transformed bya DNA coding the present enzyme, and the protein expressed in thetransformant is collected, and thereby preparing the present enzyme. Thecollected protein is treated as appropriate according to the intendeduse. The present enzyme thus obtained as a recombinant protein may besubjected to various modifications. For example, the present enzymecomposed of a recombinant protein linked to any peptide or protein canbe obtained by producing a recombinant protein using a vector into whicha DNA coding the present enzyme has been inserted together with otherappropriate DNA. In addition, modification for causing addition of asugar chain and/or a lipid, or N- or C-terminal processing may becarried out. These modifications allow, for example, extraction of arecombinant protein, simplification of purification, or addition ofbiological functions.

(Maltotriosyl transferase gene) A second aspect of the present inventionrelates to a maltotriosyl transferase gene. The gene according to oneembodiment of the present invention contains the DNA coding the aminoacid sequence set forth in SEQ ID NO: 7 or 8. A specific example of theembodiment is the DNA composed of the base sequence set forth in SEQ IDNO: 6.

In general, when a DNA coding a certain protein is partially modified,the protein coded by the modified DNA may have the equivalent functionto the protein coded by the unmodified DNA. More specifically,modification of the DNA sequence does not substantially influence thefunction of the protein coded, so that the function of the coded proteinmay be maintained before and after the modification. Therefore, anotherembodiment of the present invention provides a DNA (hereinafter, alsoreferred to as “equivalent DNA”) which has an equivalent base sequenceto the base sequence set forth in SEQ ID NO: 6, and codes a proteinhaving maltotriosyl transferase activity. The term “equivalent basesequence” in this case means a base sequence which is partiallydifferent from the nucleic acid set forth in SEQ ID NO: 6, but thedifference exerts no substantial influence on the function (in thiscase, maltotriosyl transferase activity) of the protein coded.

A specific example of the equivalent DNA is a DNA which hybridizes withthe base sequence complementary to the base sequence set forth in SEQ IDNO: 6 under stringent conditions. The term “stringent conditions” inthis case means the conditions under which a so-called specific hybridis formed, but nonspecific hybrid will not be formed. Such stringentconditions are known to those skilled in the art, and may be establishedconsulting with, for example, Molecular Cloning (Third Edition, ColdSpring Harbor Laboratory Press, New York) and Current protocols inmolecular biology (edited by Frederick M. Ausubel et al., 1987). Thestringent conditions include, for example, incubation at about 42° C. to50° C. in a hybridization solution (50% formamide, 10×SSC (0.15 M NaCl,15 mM sodium citrate, pH 7.0), 5×Denhardt solution, 1% SDS, 10% dextransulfate, 10 μg/ml modified salmon sperm DNA, and 50 mM phosphoric acidbuffer (pH 7.5)), followed by washing with 0.1×SSC and 0.1% SDS at about65° C. to 70° C. Under even more preferred stringent conditions, forexample, the hybridization solution is 50% formamide, 5×SSC (0.15 MNaCl, 15 mM sodium citrate, pH 7.0), 1×Denhardt solution, 1% SDS, 10%dextran sulfate, 10 μg/ml modified salmon sperm DNA, and 50 mMphosphoric acid buffer (pH 7.5)).

Specific examples of the equivalent DNA include a DNA which is composedof a base sequence including substituted, deletion, insertion, addition,or inversion of one or a plurality of (preferably one to several) baseswith reference to the base sequence set forth in SEQ ID NO: 6, and codesa protein having maltotriosyl transferase activity. The substitution ordeletion of the base may arise in a plurality of regions. The term “aplurality of” means, for example, from 2 to 40 bases, preferably from 2to 20 bases, and more preferably from 2 to 10 bases, though the numbervaries depending on the position and type of the amino acid residue inthe steric structure of the protein coded by the DNA. Theabove-described equivalent DNA is obtained by, for example, modifyingthe DNA having the base sequence set forth in SEQ ID NO: 6 byrestriction enzyme treatment, treatment with exonuclease or DNA ligase,or mutagenesis such as site-directed mutagenesis (Molecular Cloning,Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, NewYork) or random mutagenesis (Molecular Cloning, Third Edition, Chapter13, Cold Spring Harbor Laboratory Press, New York), so as to includesubstitution, deletion, insertion, addition, and/or inversion of thebase. Alternatively, the equivalent DNA may also be obtained by anyother method such as ultraviolet irradiation. Other examples of theequivalent DNA include DNAs having the above-described difference in thebases due to polymorphism as typified by SNP (single nucleotidepolymorphism).

The gene of the present invention may be prepared in an isolated stateusing, for example, standard genetic engineering technique, molecularbiological technique, or biochemical technique, in consultation with thesequence information disclosed in the present description or thesequence list attached thereto. Specifically, the gene may be preparedfrom the genomic DNA library or cDNA library of Geobacillus sp. APC9669,or the intracellular extract of Geobacillus sp. APC9669, using asappropriate an oligonucleotide probe primer which can specificallyhybridizes with the gene of the present invention. The oligonucleotideprobe primer is readily synthesized using, for example, a commerciallyavailable automatic DNA synthesizer. The method for constructing thelibrary used for the preparation of the gene of the present inventionmay refer to, for example, Molecular Cloning, Third Edition, Cold SpringHarbor Laboratory Press, New York.

For example, a gene having the base sequence set forth in SEQ ID NO: 6may be isolated by the hybridization method using the whole or a portionof the base sequence or its complementary sequence as the probe.Alternatively, the gene may be amplified and isolated by nucleic acidamplification reaction (for example PCR) using a syntheticoligonucleotide primer designed so as to specifically hybridize with aportion of the base sequence. Alternatively, the intended gene may beobtained by chemical synthesis, based on the information on the aminoacid sequence set forth in SEQ ID NO: 7 or the base sequence set forthin SEQ ID NO: 6 (reference: Gene, 60(1), p. 115-127 (1987)).

An example of the method for obtaining the gene of the present inventionis described below. Firstly, the present enzyme (maltotriosyltransferase) is isolated and purified from Geobacillus sp. APC9669,thereby obtaining information on the partial amino acid sequence. Thepartial amino acid sequence is determined by, for example, the purifiedmaltotriosyl transferase is directly subjected to Edman degradationaccording to an ordinary method [Journal of Biological Chemistry, vol.256, p. 7990 to 7997 (1981)], and then to amino acid sequence analysis[Protein Sequencer 476A, Applied Biosystems]. It is effective thatlimited proteolysis is carried out by the action of a proteolyticenzyme, the peptide fragment thus obtained is separated and purified,and the purified peptide fragment thus obtained is subjected to aminoacid sequence analysis.

Based on the partial amino acid sequence information thus obtained, amaltotriosyl transferase gene is cloned. For example, the cloning mayuse a hybridization method or PCR. When the hybridization method isused, for example, the method described in Molecular Cloning (ThirdEdition, Cold Spring Harbor Laboratory Press, New York) may be used.

When the PCR method is used, the following method may be used. Firstly,using the genome DNA of a microorganism producing a maltotriosyltransferase as template, PCR reaction is carried out using a syntheticoligonucleotide primer designed based on the information concerning thepartial amino acid sequence, thereby obtaining the desired genefragment. The PCR method is carried out in accordance with the methoddescribed in “PCR Technology (edited by Erlich H A, Stocktonpress,1989)”. Furthermore, when the base sequence of the amplified DNAfragment is determined using a commonly used method, such as the dideoxychain terminator method, a sequence corresponding to the partial aminoacid sequence of a maltotriosyl transferase is found in addition to thesequence of the synthetic oligonucleotide primer in the determinedsequence, and thus a part of the desired maltotriosyl transferase geneis obtained. Furthermore, the gene coding the full length of themaltotriosyl transferase can be cloned by, for example, hybridizationusing the gene fragment thus obtained as a probe.

In the below-described example, the sequence of the gene coding themaltotriosyl transferase produced by Geobacillus sp. APC9669 wasdetermined using the PCR method. The entire base sequence of the genecoding the maltotriosyl transferase derived from Geobacillus sp. APC9669is set forth in SEQ ID NO: 6. Furthermore, the amino acid sequence codedby the base sequence was determined (SEQ ID NO: 7). In addition to thebase sequence set forth in SEQ ID NO: 6, there are a plurality of basesequences which correspond to the amino acid sequence set forth in SEQID NO: 7.

Using the whole or a portion of the maltotriosyl transferase gene (SEQID NO: 6), the entire base sequence of which has been revealed, as thehybridization probe, a DNA having high homology to the maltotriosyltransferase gene set forth in SEQ ID NO: 6 can be chosen from thegenomic DNA library or cDNA library of the microorganism producing othermaltotriosyl transferase.

The primer for PCR can be designed in the same manner. The PCR reactionusing the primer allows the detection of gene fragments having highhomology to the above-described maltotriosyl transferase gene, andacquisition of the entire gene.

Whether the gene obtained codes a protein having maltotriosyltransferase activity or not may be ascertained by producing the proteinof the gene, and measuring its maltotriosyl transferase activity.Alternatively, whether the gene obtained codes a protein havingmaltotriosyl transferase activity or not may be judged by comparing thebase sequence of the gene (or the amino acid sequence coded) with thebase sequence of the above-described maltotriosyl transferase gene (orthe amino acid sequence coded), thereby determining the gene structureand homology.

The elucidation of the primary structure and gene structure allows theacquisition of a modified maltotriosyl transferase (gene includingsubstitution, deletion, insertion, addition, or inversion of one or aplurality of amino acid residues) through the introduction of randommutation or site-specific mutation. As a result of this, obtained is agene which has maltotriosyl transferase activity, but codes amaltotriosyl transferase having different properties such as optimumtemperature, stable temperature, optimum pH, stable pH, and substratespecificity. In addition, a modified maltotriosyl transferase isproduced by genetic engineering technique.

The introduction of mutation is carried out in consideration of, forexample, the characteristic sequence in the gene sequence. Theconsideration to the characteristic sequence is given by, for example,considering the prediction of the steric structure of the protein, andhomology to known proteins.

Examples of the method for introducing random mutation include chemicaltreatment of DNA such as a method of causing transition mutation forconverting cytosine base to uracil base by the action of sodiumhydrogensulfite [Proceedings of the National Academy of Sciences of theUSA, vol. 79, p. 1408 to 1412 (1982)], biochemical methods such as amethod of causing base substitution during synthesis of a double-strandin the presence of [α-S]c-1NTP [Gene, vol. 64, p. 313 to 319 (1988)],and PCR methods such as a method of carrying out PCR in a reactionsystem containing manganese, thereby decreasing the accuracy ofnucleotide uptake [Analytical Biochemistry, vol. 224, p. 347 to 353(1995)].

Examples of the method of introducing site-specific mutation include amethod of using amber mutation [gapped duplex method, Nucleic AcidsResearch, vol. 12, vol. 24, p. 9441 to 9456 (1984)], a method using therestricted enzyme recognition site [Analytical Biochemistry, vol. 200,p. 81 to 88 (1992), Gene, vol. 102, p. 67 to 70 (1991)], a method ofusing dut (dUTPase) and ung (uracil DNA glycosylase) mutation [Kunkelmethod, Proceedings of the National Academy of Sciences of the USA, vol.82, p. 488 to 492 (1985)], a method including amber mutation using DNApolymerase and DNA ligase [Oligonucleotide-directed Dual Amber: ODA,Gene, vol. 152, p. 271 to 275 (1995), Japanese Unexamined PatentApplication Publication No. 7-289262], a method of using a host whichinduced DNA repair system (Japanese Unexamined Patent ApplicationPublication No. 8-70874), a method using a protein which catalyzes DNAchain exchange reaction (Japanese Unexamined Patent ApplicationPublication No. 8-140685), a PCR method using two mutation introducingprimers having restricted enzyme recognition sites (U.S. Pat. No.5,512,463), a PCR method using a double-stranded DNA vector and twoprimers having inactivated drug resistant genes [Gene, vol. 103, p. 73to 77 (1991)], and a PCR method using amber mutation [InternationalPublication WO98/02535].

The use of a commercially available kit facilitates the introduction ofsite-specific mutation. Examples of the commercially available kitinclude Mutan (registered trademark)-G (Takara Shuzo Co., Ltd.) usingthe gapped duplex method, Mutan (registered trademark)-K (Takara ShuzoCo., Ltd.) using the Kunkel method, Mutan (registeredtrademark)-ExpressKm (Takara Shuzo Co., Ltd.) using the ODA method, andQuikChange™ Site-Directed Mutagenesis Kit (STRATAGENE) using a mutationintroducing primer and DNA polymerase derived from Pyrococcus furiosus.Examples of the kit using the PCR method include TaKaRa LA PCR in vitroMutagenesis Kit (Takara Shuzo Co., Ltd.) and Mutan (registeredtrademark)-Super Express Km (Takara Shuzo Co., Ltd.).

As described above, the present invention provides the primary structureand gene structure of a maltotriosyl transferase, and thus allowsgenetic engineering production of a protein having maltotriosyltransferase activity at a low cost, with high purity.

(Recombinant vector) Another aspect of the present invention relates toa recombinant vector containing the maltotriosyl transferase gene of thepresent invention. In the present description, the term “vector” means anucleic acid molecule capable of transporting the nucleic acid moleculeinserted therein into the target such as a cell type, and the type andform of the vector are not particularly limited. Accordingly, the vectorof the present invention may be in the form of a plasmid vector, acosmid vector, a phage vector, or a virus vector (for example, anadenovirus vector, an adeno-associated virus vector, a retrovirusvector, or a herpesvirus vector).

An appropriate vector is selected according to the intended use (cloningor protein expression), and in consideration of the type of the hostcell. Specific examples of the vector include vectors in Escherichiacoli (M13 phage or its variants, X, phage and its variants, pBR322 andits variants (for example, pB325, pAT153, and pUC8)), vectors in yeast(for example, pYepSec1, pMFa, and pYES2), vectors in insect cells (forexample, pAc and pVL), and vectors in mammalian cells (for example,pCDM8 and pMT2PC).

The recombinant vector of the present invention is preferably anexpression vector. The term “expression vector” means a vector capableof introducing the nucleic acid inserted therein into the desired cell(host cell), and expressing it in the cell. The expression vectornormally contains the promoter sequence necessary for the expression ofthe nucleic acid inserted therein, and an enhancer sequence promotingthe expression. An expression vector containing a selection marker maybe used. When the expression vector of this type is used, whether theexpression vector has been introduced or not (and the degree ofintroduction) can be ascertained using the selection marker.

The insertion of the gene of the present invention into the vector,insertion of the selection marker gene (when necessary), and insertionof promoter (when necessary) may be carried out using a standardrecombinant DNA technique (for example, a well-known method using arestriction enzyme and DNA ligase, see Molecular Cloning, Third Edition,1.84, Cold Spring Harbor Laboratory Press, New York).

(Transformant) The present invention also relates to a host cell(transformant) into which the gene of the present invention has beenintroduced. In the transformant of the present invention, the gene ofthe present invention exists as a foreign molecule. The transformant ofthe present invention is preferably prepared by transfection ortransformation using the above-described vector of the presentinvention. The transfection or transformation may be carried out by, forexample, the calcium phosphate cosedimentation method, electroporation(Potter, H. et al., Proc. Natl. Acad. Sci. U.S.A. 81, 7161-7165 (1984)),lipofection (Feigner, P. L. et al., Proc. Natl. Acad. Sci. U.S.A. 84,7413-7417 (1984)), microinjection (Graessmann, M. & Graessmann, A.,Proc. Natl. Acad. Sci. U.S.A. 73, 366-370 (1976)), Hanahan's method(Hanahan, D., J. Mol. Biol. 166, 557-580 (1983)), lithium acetate method(Schiestl, R. H. et al., Curr. Genet. 16, 339-346 (1989)), orprotoplast-polyethylene glycol method (Yelton, M. M. et al., Proc. Natl.Acad. Sci. 81, 1470-1474 (1984)).

The host cell is not particularly limited as long as the maltotriosyltransferase of the present invention is expressed, and is selected from,for example, bacteria belonging to the genus Bacillus such as Bacillussubtillus, Bacillus likemiformis, and Bacillus circulans, lactic acidbacteria such as Lactococcus, Lactobacillus, Streptococcus, Leuconostoc,and Bifidobacterium, other bacteria such as Escherichia andStreptomyces, yeasts such as Saccharomyces, Kluyveromyces, Candida,Torula, and Torulopsis, and filamentous bacteria (fungi) belonging tothe genus Aspergillus, such as Aspergillus oryzae and Aspergillus niger,and those belonging to the genus Penicillium, Trichoderma, and Fusarium.

(Method for producing maltotriosyl transferase) Yet another aspect ofthe present invention provides a method for producing a maltotriosyltransferase. One embodiment of the production method of the presentinvention includes a step of incubating a microorganism belonging to thegenus Geobacillus and having a capability to produce the present enzyme(maltotriosyl transferase) (step (1)), and a step of collecting amaltotriosyl transferase from the culture solution and/or bacterialcells after culturing (step (2)).

The microorganism belonging to the genus Geobacillus in the step (1) isnot particularly limited as long as it has a capacity of producing thepresent enzyme. For example, the above-described Geobacillus sp. APC9669may be used as the microorganism. The culture method and cultureconditions are not particularly limited as long as the desired enzyme isproduced. More specifically, on condition that the present enzyme isproduced, the culture method and conditions may be appropriatelyestablished according to the microorganism used. The culture method maybe liquid culture or solid culture, and is preferably liquid culture.Taking liquid culture as an example, the culture conditions aredescribed below.

The medium is not particularly limited as long as it is suitable forgrowing the microorganism used. Examples of the medium include thosecontaining a carbon source such as glucose, sucrose, genthiobiose,soluble starch, glycerol, dextrin, molasses, or an organic acid, inaddition, ammonium sulfate, ammonium carbonate, ammonium phosphate,ammonium acetate, or a nitrogen source such as peptone, yeast extract,corn steep liquor, casein hydrolysate, bran, or meat extract, inaddition an inorganic salt such as a potassium salt, a magnesium salt, asodium salt, a phosphate, a manganese salt, an iron salt, or a zincsalt. In order to accelerate the growth of the microorganism, a vitaminand an amino acid may be added to the medium. The pH of the medium isadjusted to, for example, about 3 to 10, and preferably about 7 to 8,and the incubation temperature is normally from about 10 to 80° C.,preferably about 30 to 65° C. The microorganism is cultured for about 1to 7 days, preferably for about 2 to 4 days under aerobic conditions.The culture method may be, for example, a shake culture method or anaerobic deep culture method using a jar fermenter.

After culturing under the above-described conditions, a maltotriosyltransferase is collected from the culture solution or bacterial cells(step (2)). When the enzyme is collected from the culture solution, forexample, the culture supernatant is subjected to, for example,filtration or centrifugation thereby removing insoluble matter, followedby separation and purification through an appropriate combination of,for example, concentration using an ultrafiltration membrane, saltingout by ammonium sulfate precipitation, dialysis, and variouschromatography procedures such as ion exchange chromatography, and thusobtaining the present enzyme.

On the other hand, when the enzyme is collected from bacterial cells,the bacterial cells are crushed by, for example, pressurization orultrasonication, and then subjected to separation and purification inthe same manner as described-above, and thus obtaining the presentenzyme. Alternatively, the bacterial cells may be collected in advancefrom the culture solution by, for example, filtration or centrifugation,and then the above-described procedure (crushing of bacterial cells,separation, and purification) may be carried out.

Confirmation of expression and identification of the expression productare readily achieved using an antibody against the maltotriosyltransferase. Alternatively, the expression may be confirmed by measuringthe maltotriosyl transferase activity.

According to another embodiment of the present invention, a maltotriosyltransferase is produced using the above-described transformant. In theproduction method according to this embodiment, firstly, thetransformant is cultured under the conditions suitable for theproduction of the protein to be coded by the gene introduced into thetransformant (step (i)). Culture conditions for transformants containingvarious vector hosts are known, and those skilled in the art can readilyestablish appropriate culture conditions. Following the culturing step,the produced protein (more specifically maltotriosyl transferase) iscollected (step (ii)). The collection and subsequent purification arecarried out in the same manner as in the above-described embodiment.

The degree of purification of the present enzyme is not particularlylimited. In addition, the final form may be liquid or solid (includingpowder).

(Enzyme product) The enzyme of the present invention may be provided inthe form of an enzyme product. The enzyme product may contain, inaddition to the active ingredient (the enzyme of the present invention),an excipient, a buffer, a suspending agent, a stabilizer, apreservative, an antiseptic, or a normal saline solution. Examples ofthe excipient include starch, dextrin, maltose, trehalose, lactose,D-glucose, sorbitol, D-mannitol, white sugar, and glycerol. Examples ofthe buffer include phosphates, citrates, and acetates. Examples of thestabilizer include propylene glycol and ascorbic acid. Examples of thepreservative include phenol, benzalkonium chloride, benzyl alcohol,chlorobutanol, and methylparaben. Examples of the antiseptic includeethanol, benzalkonium chloride, paraoxybenzoic acid, and chlorobutanol.

(Use of maltotriosyl transferase) Another aspect of the presentinvention is to provide a food production and processing method as a useof the maltotriosyl transferase (the present enzyme). According to thefood production and processing method of the present invention, thepresent enzyme is acted on a food or food ingredient containing apolysaccharide and/or an oligosaccharide having α-1,4 glucoside bonds,thereby improving the function of the food. Examples of the food includebread, rice, and rice cake. Examples of the food ingredient includevarious ingredients containing starch, amylose, amylopectin, andmaltooligosaccharide. The purity of the ingredient is not particularlylimited. The present enzyme may be acted on the ingredient mixed withother substance. Alternatively, the present enzyme may be acted on twoor more ingredients at the same time.

Examples

<Measurement of maltotriosyl transferase activity> The activity of themaltotriosyl transferase was measured as follows. More specifically, 0.5mL of an enzyme solution was added to 2 mL of a 10 mmol/L MES buffersolution (pH 6.5) containing 1% maltotetraose (Hayashibara BiochemicalLaboratories, Inc.), and allowed to stand at 40° C. for 60 minutes.After the standing, the mixture was heated for 5 minutes in a boilingwater bath, and then cooled in running water. The amount of glucose thusproduced was quantified by Glucose CH-test WAKO (Wako Pure ChemicalIndustries, Ltd.). Under the present conditions, the amount of theenzyme producing 1 mmol of glucose in 2.5 mL of the reaction solutionfor 1 minute was set at 1 unit.

<Confirmation of maltotriosyl transferase activity> The activity of themaltotriosyl transferase was confirmed as follows, together with theabove-described <Measurement of maltotriosyl transferase activity>. Morespecifically, 15 μL of a 1.0 u/mL enzyme solution was added to 985 μL ofa 5 mmol/L acetic acid buffer solution (pH 6.0) containing 10.3 mmol/Lmaltotetraose (Hayashibara Biochemical Laboratories, Inc.), and allowedto stand for 1, 2, or 3 hours at 50° C. After the standing, the mixturewas heated in a boiling water bath for 5 minutes, and then cooled inrunning water. The cooled reaction solution was desalted as needed usinga cationic resin and an anionic resin, and the reaction solution wasanalyzed by HPLC. The HPLC apparatus was “Prominence UFLC” manufacturedby Shimadzu Co., Ltd., the column was “MC1 GEL CK04S” manufactured byMitsubishi Chemical Corporation, the eluent was water at a flow rate of0.4 mL/minute, and the detection was carried out using a differentialrefractometer. The area percentages of the substrate and productobtained were converted to molar quantities, and the consumption rateand production rate were calculated. When a purified maltotriosyltransferase was analyzed, for example, the ratio of the production ratewas, for example, heptasaccharide:trisaccharide=about 92:about 8.

1. Production and purification of maltotriosyl transferase derived fromGeobacillus sp. APC9669 Geobacillus sp. APC9669 was cultured undershaking at 45° C. for 2 days using the liquid medium having thecomposition shown in Table 1. The culture supernatant thus obtained wasconcentrated by five folds using a UF membrane (AIP-1013D, Asahi KaseiCorporation), and then ammonium sulfate was added to make a 50%saturated solution. The precipitate fraction was redissolved in a 20mmol/L tris-hydrochloride buffer (pH 8.0) containing 5 mmol/L calciumchloride, and then ammonium sulfate was added to give a finalconcentration of 0.5 mol/L. After removing the precipitate thus formedby centrifugation, the solution was passed through HiLoad 26/10 PhenylSepharose HP column (GE Healthcare) which had been equilibrated with a20 mmol/L tris-hydrochloride buffer (pH 8.0) containing 0.5 mol/Lammonium sulfate and 5 mmol/L calcium chloride, and the adsorbedmaltotriosyl transferase protein was eluted by ammonium sulfate linearconcentration gradient from 0.5 mol/L to 0 mol/L.

TABLE 1 Maltotriosyl transferase producing medium (w/v) Yeast extract1.5% Soybean peptone 0.5% Sodium chloride 0.5% Soluble starch 0.4%

The collected maltotriosyl transferase active fraction was concentratedusing a UF membrane, and the buffer was replaced with a 20 mmol/Ltris-hydrochloride buffer (pH 8.0) containing 5 mmol/L calcium chloride.The sample in the replaced buffer was passed through HiLoad 26/10 QSepharose HP column (GE Healthcare) which had been equilibrated with a20 mmol/L tris-hydrochloride buffer (pH 8.0) containing 5 mmol/L calciumchloride, and the adsorbed maltotriosyl transferase protein was elutedby NaCl linear concentration gradient from 0 mol/L to 1 mol/L.

Furthermore, the collected maltotriosyl transferase active fraction wasconcentrated using a UF membrane, and then the buffer was replaced witha 50 mM phosphate buffer solution (pH 7.2) containing 0.15 M NaCl. Thesolution was passed through HiLoad 26/60 Superdex 200 pg column (GEHealthcare) which had been equilibrated with a 50 mM phosphate buffersolution (pH 7.2) containing 0.15 M NaCl, and eluted by the buffersolution. The maltotriosyl transferase active fraction was collected,and desalted and concentrated using an ultrafiltration membrane, andthus obtaining a purified enzyme preparation. The purified enzyme wassubjected to the following evaluation of various properties.

The results of purification in the respective steps are shown in Table2. The specific activity in the final stage was about 41 times that ofthe crude enzyme. FIG. 5 shows the result of SDS-PAGE (CBB staining) ina 10-20% gradient gel carried out on the samples in the respective stepsof the purification process. The result indicates that the purifiedenzyme preparation (Lane 2) is a single protein in the SDS-PAGE.

TABLE 2 Total amount Total Specific of protein activity activityRecovery (mg) (U) (u/mg) rate (%) Concentrate 42 300 3.9 100 Ammonium8.5 220 22.9 73 sulfate fraction Phenyl HP 0.50 58 100 19 Q HP 0.37 53139 18 Superdex200 0.12 19 158 6.3

2. Properties of maltotriosyl transferase (1) Optimum reactiontemperature In accordance with the above-described maltotriosyltransferase activity measurement method, reaction was carried out atreaction temperatures of 30° C., 40° C., 45° C., 50° C., 55° C., 60° C.,65° C., 70° C., and 75° C. The results were expressed as relativeactivity, taking the value at the temperature at which the highestactivity was exhibited as 100%. The optimum reaction temperature was inthe vicinity of 50° C. (FIG. 1).

(2) Optimum reaction pH In accordance with the above-describedmaltotriosyl transferase activity measurement method, the measurementwas carried out under reaction conditions at 40° C. for 60 minutes inbuffer solutions (universal buffer solutions at pH 4.0, pH 4.5, pH 5.0,pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 9.0, pH 10.0, and pH11.0). The results were expressed as relative activity, taking the valueat the pH at which the highest activity was exhibited as 100%. Theoptimum reaction pH was in the vicinity of about 7.5 (FIG. 2).

(3) Thermostability A 6 u/mL enzyme solution was heat treated for 30minutes at 30° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70°C., or 75° C. in a 10 mmol/L MES buffer solution (pH 6.5), and thenresidual activity was measured in accordance with the above-describedmaltotriosyl transferase activity measurement method. The results wereexpressed as residual activity, taking the activity of the sampleunheated as 100%. After heat treatment at 65° C. for 30 minutes, theresidual activity was 90% or more, and the activity was stable up to 65°C. (FIG. 3).

(4) pH stability A 6 u/mL enzyme solution was treated at 40° C. for 30minutes in a buffer solution (universal buffer solution at pH 3.0, pH4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH9.0, pH 10.0, or pH 11.0), and then the activity was measured inaccordance with the above-described maltotriosyl transferase activitymeasurement method. In the range from pH 5.0 to pH 10.0, the residualactivity was 85% or more, and the activity was stable in the range frompH 5.0 to pH 10.0 (FIG. 4).

(5) Molecular weight measurement by SDS-PAGE SDS-PAGE was carried out inaccordance with by the method of Laemmli et al. The molecular weightmarker used herein was Low Molecular Weight Calibration Kit forElectrophoresis (GE Healthcare), which contained Phosphorylase b (97,000Da), Albumin (66,000 Da), Ovalbumin (45,000 Da), Carbonic anhydrase(30,000 Da), Trypsin inhibitor (20,100 Da), or α-Lactalbumin (14,400 Da)as the standard protein. Electrophoresis was carried out at 20 mA/gelfor about 80 minutes using a gradient gel at a gel concentration of10-20% (Wako Pure Chemical Industries, Ltd.), and the molecular weightwas determined; the molecular weight was about 83 kDa (FIG. 5).

(6) Isoelectric point The isoelectric point of the present enzyme wasabout 4.5, as measured by isoelectric focusing (600V, 4° C., 48 hours)using Ampholine.

(7) Substrate specificity The maltotriosyl transferase activity fordifferent substrates was tested. a) Substrate specificity tomaltooligosaccharide Substrate specificity to maltooligosaccharides wastested by the following method. The enzyme was added to 10 mmol/Lmaltooligosaccharides so as to give a concentration of 0.002 u/mL, andallowed to stand at 50° C. for 1, 2, or 3 hours. After the standing, themixture was heated in a boiling water bath for 5 minutes, and thencooled in running water. The cooled reaction solution was desalted asneeded using a cationic resin and an anionic resin, and the reactionsolution was analyzed by HPLC. The HPLC apparatus was “Prominence UFLC”manufactured by Shimadzu Co., Ltd., the column was “MCI GEL CK04S”manufactured by Mitsubishi Chemical Corporation, the eluent was water ata flow rate of 0.4 mL/minute, and the detection used a differentialrefractometer. The area percentages of the substrate and productobtained were converted to molar quantities, and the consumption rateand production rate were calculated. The reaction rates for therespective maltooligosaccharides were calculated as follows. The ratefor maltotetraose was the sum of the heptasaccharide and trisaccharideproduction rates. The rate for maltopentaose was the sum of theoctasaccharide and trisaccharide production rates. The rate formaltohexaose was calculated by halving the difference between thetrisaccharide and nonasaccharide production rates, and then adding thevalue to the nonasaccharide production rate.

TABLE 3 Substrate Relative rate (%) Maltose 0 Maltotriose 0Maltotetraose 83 Maltopentaose 100 Maltohexaose 89

No reaction product was observed for maltose and maltotriose. Highactivity was exhibited for maltotetraose, maltopentaose, andmaltohexaose.

b) Substrate specificity to polysaccharides Substrate specificity tocyclodextrin, soluble starch, amylose, and amylopectin was tested by thefollowing method. The enzyme was added to 10 mmol/Lmaltooligosaccharides so as to give a concentration of 0.002 u/mL, andallowed to stand at 50° C. for 1, 2, or 3 hours. After the standing, themixture was heated in a boiling water bath for 5 minutes, and thencooled in running water. To 200 μL of the solution, Rhizopus-derivedglucoamylase (Wako Pure Chemical Industries, Ltd.) was added in aconcentration of 0.03 mg in 1.0 unit, and allowed to stand at 50° C.overnight. After the standing, the mixture was heated in a boiling waterbath for 5 minutes, and then cooled in running water. The cooledreaction solution was desalted as needed using a cationic resin and ananionic resin, and the reaction solution was analyzed by HPLC. The HPLCapparatus was “Prominence UFLC” manufactured by Shimadzu Co., Ltd., thecolumn was “MCI GEL CK04S” manufactured by Mitsubishi ChemicalCorporation, the eluent was water at a flow rate of 0.4 mL/minute, andthe detection used a differential refractometer. When the sample treatedwith the enzyme (maltotriosyl transferase) showed a time-dependentincrease of the peak of trisaccharide or higher saccharide in comparisonwith the untreated sample, the reaction product was judged as present(+), while no increase was observed, the reaction product was judged asabsent (−).

TABLE 4 Substrate Presence or absence of product α-cyclodextrin −β-cyclodextrin − γ-cyclodextrin − Amylose + Amylopectin + Soluble starch+

For cyclodextrin, no reaction product was observed. For soluble starch,amylose, and amylopectin, time-dependent increases were found in thepeaks of trisaccharide or higher saccharide. These polysaccharides werefound to serve as substrates. The fact that glucoamylase hydrolyzesα-1,4 and α-1,6 bonds indicates that transglycosylation products wereformed also for other binding modes.

(8) Influence of substrate concentration on enzyme reaction product Theinfluence of the substrate concentration on the enzyme reaction productwas studied using maltotetraose as substrate. The enzyme was added to0.67, 1.0, 3.0, 10, 30, or 70% (w/v) maltotetraose in such a manner thatthe maltotetraose residue is 85% or more after reaction for 3 hours, andallowed to stand at 50° C. for 1, 2, or 3 hours. After the standing, themixture was heated in a boiling water for 5 minutes, and cooled inrunning water. The cooled reaction solution was desalted as needed usinga cationic resin and an anionic resin, and the reaction solution wasanalyzed using HPLC. The HPLC apparatus was “Prominence UFLC”manufactured by Shimadzu Co., Ltd., the column was “MCI GEL CK04S”manufactured by Mitsubishi Chemical Corporation, the eluent was water ata flow rate of 0.4 mL/minute, and the detection used a differentialrefractometer. The area percentages of the substrate and productobtained were converted to molar quantities, and the production rate wascalculated. The results indicate that the transglucosylation was 90% ormore under all the substrate concentration conditions (from 0.67 to 70%(w/v)).

TABLE 5 Substrate Reaction production rate (molar ratio) concentrationTransfer product Decomposition product (% (w/v)) (heptasaccharide)(trisaccharide) 0.67  92% 8% 1.0  96% 4% 3.0 100% 0% 10 100% 0% 30 100%0% 70 100% 0%

3. Bread baking The maltotriosyl transferase was added to bread dough,and baked into bread. Basic ingredients of English loaf (strong flour260 g; sugar 13 g; salt 5.2 g; shortening 10.4 g; L-ascorbic acid 0.013g; cold water 192 g; and dry yeast 3.1 g) or the mixture of the basicingredients and 120 u of the maltotriosyl transferase was charged intoNational Automatic Home Bakery SD-BT150 (Panasonic Corporation). Afterbaking, the bread was allowed to cool at 26° C. for 1 hour, subsequentlythe bread was placed in a plastic bag so as to prevent moistureevaporation, and stored at 26° C. After storage for 1 or 5 days, thebread was sliced into 2 cm thick pieces, and the central portion of thebread was cut into a column having a diameter of 47 mm. The hardness ofthe bread was determined by measuring the maximum load when the breadwas compressed by 1.5 cm at a compression speed of 2 mm/minute, usingFUDOH rheometer NRM-2002J (Sun Scientific Co., Ltd., Rheotech). Theresults are shown in FIG. 6. The hardness of the bread stored for fivedays was compared between the enzyme-containing and enzyme-free samples,taking the hardness of the bread after storage for one day as 100%. Thehardness of the enzyme-containing sample was 125%, indicating thathardening of the bread was suppressed and softness was maintained incomparison with those of the enzyme-free sample (hardness: 207%).

4. Rice cooking 75 g of rice was washed with water, to which 150 mL ofwater was added alone or together with 40 u of the maltotriosyltransferase, the mixture was allowed to stand for 2 hours at roomtemperature, and then cooked by an ordinary procedure to obtain cookedrice. The cooked rice was stored at 4° C. for 7 days. The degree ofgelatinization before and after storage was measured by the BAP method.The degree of gelatinization of the enzyme-containing sample was 96.6%immediately after rice cooking, and 69.5% after 7 days as measured bythe BAP method (Table 6). On the other hand, the degree ofgelatinization of the enzyme-free sample was 95.3% immediately afterrice cooking, and 59.7% after 7 days. In the enzyme-containing sample,deterioration of the degree of gelatinization was suppressed, orretrogradation of starch was suppressed.

TABLE 6 Degree of gelatinization After 1 day After 7 daysEnzyme-containing sample 96.6% 69.5% Enzyme-free sample 95.3% 59.7%

5. Production of rice cake 200 g of rice powder was mixed with 165 g ofwater, and steamed with water vapor for 15 minutes. Subsequently, thesteamed powder was stirred in a mixer (Kitchen Aid KSM5, FMICorporation). When the temperature of the dough reached about 65° C., 30u of the maltotriosyl transferase was added to and mixed to make anenzyme-containing sample. The sample was molded in a plastic petri dish,allowed to cool, and stored at 15° C. After storage for 24 hours, therice cake was sliced into 10 mm thick pieces, and the central portion ofthe rice cake was cut into a column having a diameter of 25 mm. Thehardness of the rice cake was determined by measuring the maximum loadwhen the rice cake was compressed by 5 mm at a compression speed of 2mm/minute, using FUDOH rheometer NRM-2002J (Sun Scientific Co., Ltd.,Rheotech). The hardness of the rice cake was compared, taking thehardness of the enzyme-free rice cake after storage for 24 hours as100%. In addition, stickiness of the rice cake was also tested. Thehardness of the enzyme-containing sample was 35%, indicating thathardening of the rice cake was suppressed, and softness was maintained(Table 7). In addition, the rice cake had no stickiness.

TABLE 7 Hardness Stickiness Enzyme-containing sample  35% NoneEnzyme-free sample 100% None

6. Acquisition of gene fragment coding maltotriosyl transferase derivedfrom Geobacillus sp. APC9669 (a) Isolation of chromosomal DNA Achromosomal DNA was prepared from the bacterial cells of Geobacillus sp.APC9669 by the Saito and Miura's method (Non-Patent Document 5).

(b) Determination of partial amino acid sequence The purified sample ofthe maltotriosyl transferase obtained in 1. was subjected to the aminoacid sequence analysis, and the N-terminal 10-residue amino acidsequence (SEQ ID NO: 1) and internal peptide amino acid sequence (SEQ IDNOs: 2 and 3) were determined. (c) Preparation of DNA probe by PCR Twomixed oligonucleotides (SEQ ID NOs: 4 and 5) were synthesized based onthe N-terminal amino acid sequence and internal amino acid sequence, andused as PCR primers. Using these primers, PCR reaction was carried outunder the following conditions, wherein the template was the chromosomalDNA of Geobacillus sp. APC9669. <PCR reaction solution>

10×PCR reaction buffer solution (Takara Bio Inc.) 5.0 μl dNTP mixedsolution (2.5 mM, Takara Bio Inc.) 8.0 μl 25 mM MgCl₂ 5.0 μl 50 μM senseprimer 0.5 μl 50 μM antisense primer 0.5 μl Distilled water 29.5 μlChromosomal DNA solution (100 μg/ml) 1.0 μl LA Taq DNA polymerase(Takara Bio Inc.) 0.5 μl

<PCR reaction conditions> Stage 1: denaturation (95° C., 5 minutes) 1cycle Stage 2: denaturation (95° C., 1 minute) 30 cycles Annealing (50°C., 1 minute) Extension (72° C., 1 minute) Stage 3: extention (72° C.,10 minutes) 1 cycle

About 1.1 kb of DNA fragment thus obtained was cloned into pGEM-Teasy(Promega K.K.), and then the base sequence was confirmed; the basesequence coding the above-described partial amino acid sequence wasfound immediately after the sense primer and immediately before theantisense primer. This DNA fragment was used as the DNA probe forcloning the full length gene.

(d) Construction of gene library As a result of the southernhybridization analysis of the chromosomal DNA of Geobacillus sp.APC9669, a single band of about 5.2 kb hybridizing with the probe DNAwas found in the EcoRI digestion product. In order to clone the EcoRIDNA fragment of about 5.2 kb, a gene library was constructed as follows.The chromosomal DNA prepared in the above-described (a) was subjected toEcoRI treatment. 50 μg of the chromosomal DNA, 40 μl of 10×H buffersolution, 342.0 μl of distilled water, and 8.0 μl of EcoRI were mixed,and treated at 37° C. for 15 hours. The digestion product thus obtainedwas ligated into the EcoRI-treated pBluescript II KS+ vector(Stratagene), and thus obtaining a gene library.

(e) Screening of gene library The DNA fragment of 1.1 kb obtained in theabove-described (c) was labeled using DIG-High Prime (Roche). Using thisas DNA probe, the gene library obtained in (d) was screened by colonyhybridization. A pBlue-SAS plasmid was obtained from the positive colonythus obtained.

(f) Determination of base sequence The base sequence of the pBlue-SASplasmid was determined by an ordinary procedure. The base sequence (2304bp) coding the maltotriosyl transferase derived from Geobacillus sp.APC9669 is set forth in SEQ ID NO: 6. In addition, the amino acidsequence (767 amino acids) coded by SEQ ID NO: 6 is set forth in SEQ IDNO: 7. In the amino acid sequence, the N-terminal region amino acidsequence (SEQ ID NO: 1) determined in (b) and the internal amino acidsequences (SEQ ID NOs: 2 and 3) were found. The amino acid sequenceexcluding the signal peptide from the amino acid sequence set forth inSEQ ID NO: 7 is set forth in SEQ ID NO: 8.

7. Expression of maltotriosyl transferase derived from Geobacillus sp.APC9669 in Escherichia coli (a) Construction of plasmid expressingmaltotriosyl transferase in Escherichia coli Two oligonucleotides (SEQID NOs: 9 and 10) were synthesized based on the DNA sequences coding theN-terminal region amino acid sequence and C-terminal region amino acidsequence, and used as PCR primers. The sense primer contained the SacIrestriction enzyme recognition site, and the antisense primer containedthe XbaI restriction enzyme recognition site. Using these primers andthe chromosomal DNA having a maltotriosyl transferase gene as templates,PCR reaction was carried out under the following conditions. <PCRreaction solution>10×PCR reaction buffer solution (TOYOBO) 5.0 μl dNTPmixed solution (respectively 2.5 mM, TOYOBO) 5.0 μl 10 μM sense primer1.5 μl 10 μM antisense primer 1.5 μl 25 mM MgSO₄ 2.0 μl Distilled water33.0 μl Chromosomal DNA solution (200 μg/ml) 1.0 μl KOD-Plus-DNApolymerase (TOYOBO) 1.0 μl <PCR reaction conditions> Stage 1:denaturation (94° C., 2 minutes) 1 cycle Stage 2: denaturation (94° C.,15 seconds) 30 cycles Annealing (50° C., 30 seconds) Extension (68° C.,2 minutes 30 seconds)

The PCR product thus obtained was confirmed by electrophoresis, anddesalted by ethanol precipitation (84 μl). Subsequently, 10 μl of a 10×Mbuffer solution, 3 μl of SacI, and 3 μl of ×baI were added, andsubjected to enzyme treatment at 37° C. for 15 hours. The solutiontreated with the restriction enzyme was confirmed by electrophoresis,purified by NucleoSpin Extract II (Nippon Genetics Co., Ltd.), and thenligated into pColdII DNA vector (Takara Bio Inc.), which had beentreated with SacI and XbaI, and thus obtaining a expression plasmidpColdII-SAS.

(b) Expression of maltotriosyl transferase in Escherichia coli Theexpression plasmid pColdII-SAS was introduced into Escherichia coliJM109 Competent Cells (Takara Bio Inc.). From the transformants obtainedas ampicillin-resistant strains, the strains having pColdII-SAS intowhich the intended maltotriosyl transferase gene had been inserted wereselected by colony PCR. In addition, the transformant of Escherichiacoli JM109 having the expression vector pColdII DNA was also obtained ascontrol. These transformants were inoculated in 1 ml of the LB mediumcontaining 100 μg/ml of ampicillin, and cultured until O.D 600 reached0.4-1.0 at 37° C., 170 rpm (preculture). Subsequently, 300 μl of thepreculture solution was inoculated in 9 ml of the LB medium containing100 μg/ml of ampicillin, and cultured until O.D 600 reached 0.4-1.0 at37° C., 170 rpm. After the standing at 15° C. for 30 minutes, 9 μl of0.1 M IPTG was added, cultured at 15° C., 160 rpm for 24 hours (mainculture), and the bacterial cells were collected. The bacterial cellswere suspended in 1.0 ml of 100 mM Tris-HCl buffer (pH 6.5), 0.50 g ofglass beads having a diameter of 0.1 mm was added, and the bacterialcells were crushed using Multi-Beads Shocker (Yasui Kikai Corporation).The crushing was achieved by repeating a cycle of 120 seconds on and 60seconds of 3.75 times. The cell free-extract thus obtained was subjectedto centrifugation, and thus obtaining a soluble component.

(c) Confirmation of maltotriosyl transferase expression The solublecomponent thus obtained was subjected to SDS-PAGE. The electrophoreticapparatus was PhastSystem (GE Healthcare), and the separating gel wasPhastGel Homogeneous 7.5 (GE Healthcare). As a result of this, as shownin FIG. 7, pColdII-SAS showed significant production of a protein whichappears to be a maltotriosyl transferase in the vicinity of 83 kDa. ThepColdII DNA as control showed no corresponding protein production, sothat the protein was considered to be caused by the introduction of themaltotriosyl transferase gene (FIG. 7).

The same samples were measured for the activity in the same manner asthe above-described maltotriosyl transferase activity measurementmethod. The results are shown in Table 8.

TABLE 8 Activity Protein Specific (U/ml) (mg/ml) activity (U/mg)pColdII-SAS 44.3 0.515 86.1 pColdII 0.02 0.875 0.02

Maltotriosyl transferase activity was apparently detected in comparisonwith the control, and thus the expression of the intended maltotriosyltransferase was confirmed.

INDUSTRIAL APPLICABILITY

The maltotriosyl transferase of the present invention exhibits markedheat resistance, and thus is suitable for applications involvingreactions at high temperatures. The use of the maltotriosyl transferaseof the present invention allows enzyme reaction in high temperatureenvironments where the risk of contamination is low. In addition, whenthe maltotriosyl transferase is acted on a starch-containing food,retrogradation of starch is suppressed. Accordingly, the maltotriosyltransferase of the present invention is particularly useful in foodprocessing.

The present invention will not be limited to the description of theembodiments and examples of the present invention. Various modificationsreadily made by those skilled in the art are also included in thepresent invention, without departing from the scope of claims.

The contents of the articles, unexamined patent publications, and patentapplications specified herein are hereby incorporated herein byreference. Sequence list free text

SEQ ID NOs: 4, 5, 9, and 10: explanation of artificial sequence: primer

1-19. (canceled)
 20. A use of a maltotriosyl transferase for producingand processing a food containing a polysaccharide or oligosaccharidehaving α-1,4 glucoside bonds.
 21. The use according to claim 20, whereinthe maltotriosyl transferase is an enzyme which acts on polysaccharidesand oligosaccharides having α-1,4 glucoside bonds to transfermaltotriose units to saccharides, the maltotriosyl transferase acting onmaltotetraose as substrate to give a ratio between the maltoheptaoseproduction rate and maltotriose production rate of 9:1 to 10:0 at anysubstrate concentration ranging from 0.67 to 70% (w/v).
 22. The useaccording to claim 20, wherein the maltotriosyl transferase is an enzymederived from a microorganism.
 23. The use according to claim 20, whereinthe maltotriosyl transferase is an enzyme derived from a microorganismbelonging to the genus Geobacillus.
 24. The use according to claim 23,wherein the microorganism belonging to the genus Geobacillus isGeobacillus sp. APC9669 (accession number NITE BP-770).
 25. The useaccording to claim 20, wherein the maltotriosyl transferase comprisesthe following enzymatic chemical properties: (1) action: acts onpolysaccharides and oligosaccharides having α-1,4 glucoside bonds as abinding mode to transfer maltotriose units to saccharides; (2) substratespecificity: acts on soluble starch, amylose, amylopectin,maltotetraose, maltopentaose, and maltohexaose, while does not act onα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, maltotriose, andmaltose; and (3) molecular weight: about 83,000 (SDS-PAGE).
 26. The useaccording to claim 20, wherein the maltotriosyl transferase comprisesthe amino acid sequence set forth in SEQ ID NO: 8.